Somatic inflammatory gene mutations in human ulcerative colitis epithelium.

With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations1-7. However, whether such clonal expansion exists in the non-neoplastic intestine remains unknown. Here, using whole-exome sequencing data from 76 clonal human colon organoids, we identify a unique pattern of somatic mutagenesis in the inflamed epithelium of patients with ulcerative colitis. The affected epithelium accumulates somatic mutations in multiple genes that are related to IL-17 signalling-including NFKBIZ, ZC3H12A and PIGR, which are genes that are rarely affected in colon cancer. Targeted sequencing validates the pervasive spread of mutations that are related to IL-17 signalling. Unbiased CRISPR-based knockout screening in colon organoids reveals that the mutations confer resistance to the pro-apoptotic response that is induced by IL-17A. Some of these genetic mutations are known to exacerbate experimental colitis in mice8-11, and somatic mutagenesis in human colon epithelium may be causally linked to the inflammatory process. Our findings highlight a genetic landscape that adapts to a hostile microenvironment, and demonstrate its potential contribution to the pathogenesis of ulcerative colitis.

Reconstruction of the Human Colon Epithelium In Vivo.

Genetic lineage tracing has revealed that Lgr5+ murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs in vivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium in vivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer an LGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5+ CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5+ CoSCs, human LGR5+ CoSCs were slow-cycling in vivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs in vivo, with potential therapeutic applications in regenerative medicine.

Visualization and targeting of LGR5+ human colon cancer stem cells.

The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+ CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+ CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.

A Colorectal Tumor Organoid Library Demonstrates Progressive Loss of Niche Factor Requirements during Tumorigenesis.

Colorectal tumor is a heterogeneous disease, with varying clinical presentation and prognosis in patients. To establish a platform encompassing this diversity, we generated 55 colorectal tumor organoid lines from a range of histological subtypes and clinical stages, including rare subtypes. Each line was defined by gene expression signatures and optimized for organoid culture according to niche factor requirements. In vitro and in xenografts, the organoids reproduced the histopathological grade and differentiation capacity of their parental tumors. Notably, we found that niche-independent growth is predominantly associated with the adenoma-carcinoma transition reflecting accumulation of multiple mutations. For matched pairs of primary and metastatic organoids, which had similar genetic profiles and niche factor requirements, the metastasis-derived organoids exhibited higher metastatic capacity. These observations underscore the importance of genotype-phenotype analyses at a single-patient level and the value of our resource to provide insights into colorectal tumorigenesis and patient-centered therapeutic development.

Mini-gut organoids: reconstitution of the stem cell niche.

In the adult mammalian body, self-renewal of tissue stem cells is regulated by extracellular niche environments in response to the demands of tissue organization. Intestinal stem cells expressing Lgr5 constantly self-renew in their specific niche at the crypt bottom to maintain rapid turnover of the epithelium. Niche-regulated stem cell self-renewal is perturbed in several mouse genetic models and during human tumorigenesis, suggesting roles for EGF, Wnt, BMP/TGF-ß, and Notch signaling. In vitro niche reconstitution capitalizing on this knowledge has enabled the growth of single intestinal stem cells into mini-gut epithelial organoids comprising Lgr5(+) stem cells and all types of differentiated lineages. The mini-gut organoid culture platform is applicable to various types of digestive tissue epithelium from multiple species. The mechanism of self-renewal in organoids provides novel insights for organogenesis, regenerative medicine, and tumorigenesis of the digestive system.

Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.

Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-ß, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.

Characterization of intestinal and hepatic P450 enzymes in cynomolgus monkeys with typical substrates and inhibitors for human P450 enzymes.

Cynomolgus monkeys are widely used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Characterization of cynomolgus monkey P450s such as the mRNA expression level, substrate specificity, and inhibitor selectivity were conducted to provide helpful information in designing monkey in vivo studies and monkey-to-human extrapolation. The expression levels of 12 monkey P450 mRNAs, which are considered to be important P450 subfamilies in drug metabolism, were investigated in the liver, small intestine (duodenum, jejunum, and ileum), and colon of individual monkeys. 3. In vitro activities and intrinsic clearance values were determined in monkey intestinal and liver microsomes (MIM and MLM, respectively) using nine typical oxidative reactions for human P450s. Paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, and S-mephenytoin 4'-hydroxylation showed low activities in MIM and MLM. IC50 values of eight selective inhibitors of human P450s were determined in MIM and MLM. Inhibitory effects of furafylline and sulfaphenazole were weak in monkeys on phenacetin O-deethylation and diclofenac 4'-hydroxylation, respectively. These results show profiles of monkey P450s in both the intestine and liver in detail and contribute to a better understanding of the species difference in substrate specificity and inhibitor selectivity between cynomolgus monkeys and humans.

A nuclear receptor, hepatocyte nuclear factor 4, differently contributes to the human and mouse angiotensinogen promoter activities.

Angiotensinogen (AGT), mainly produced in the liver, is the precursor of angiotensin II, an important regulator of blood pressure and electrolyte homeostasis. We previously showed, in hepatoma-derived HepG2 cells that a hepatocyte nuclear factor 4 (HNF4) potentiated human AGT (hAGT) promoter activity and identified its binding sites (termed regions C and J) in the hAGT promoter region. We also showed in transgenic mouse (TgM) that the hAGT is abundantly expressed in the kidney where the level of endogenous mouse AGT (mAGT) expression is low. To elucidate molecular mechanisms of the AGT gene activation in the kidney, we first investigated the HNF4 and AGT expression in the mouse kidney. Northern blot, in situ hybridization and immunohistochemical analyses revealed that the hAGT and HNF4 were both expressed in the proximal tubular (PT) cells of the kidney. We then transfected the hAGT reporter constructs into immortalized mouse PT (mProx) cells and found that regions C and J contributed additively to the HNF4-potentiated hAGT promoter activity. Curiously, no obvious HNF4 binding motif was found in the corresponding region of the mAGT promoter and co-transfected HNF4 failed to activate this promoter in neither HepG2 nor mProx cells. These results suggest that the high-level hAGT expression in the TgM kidney is, at least in part, due to a presence of high-affinity HNF4 binding sites in its promoter.

Finb, a multiple zinc finger protein, represses transcription of the human angiotensinogen gene.

We previously identified a regulatory element at the 3'-downstream region of the human angiotensinogen (hANG) gene. Using this element as a probe by the Southwestern screening, we isolated a cDNA clone, encoding Finb, a transcriptional activator with multiple zinc finger domains. The N-terminal zinc finger domain of Finb bound to the GGATGG sequence within the regulatory element. Unexpectedly, Finb repressed transcription dependent on the regulatory element. Inspection of the 5'-flanking region in the hANG promoter identified the GGATGG-like elements, which prompted us to examine the effect of Finb on the hANG promoter activity. We also found the two Finb binding elements in the 5'-flanking region of the hANG gene by the gel shift assay, both of which were necessary for transcriptional repression of the hANG promoter. These findings suggest that Finb functions as a sequence-specific transcriptional repressor of the hANG gene.